The phosphoinositide 3-kinase (PI 3-kinase, PI3K) pathway transduces signals critical for lymphocyte function. PI3K generates the phospholipid PIP3 at the plasma membrane to recruit proteins that contain pleckstrin homology (PH) domains. The PH domain is an evolutionarily conserved structural fold found in proteins expressed in organisms ranging from yeast to mammals. The core of the PH domain is a 7-strand ?-barrel that is encoded by approximately 120 amino acids and is composed of two anti-parallel ? sheets and a C-terminal ? helix. The mammalian genome encodes roughly 300 PH domains found in proteins that perform diverse functions including cellular activation, cytoskeletal reorganization, vesicular trafficking, and cell cycle control. Approximately 15% of PH domains bind to phosphoinositides with high specificity and affinity (Kd: nM - low ?M range). PH domains generally interact with phosphoinositides through positively charged lysine and arginine residues within the basic motif KXn(K/R)XR. However, not all PH domains bind to PIP3. Several PH domains interact with phosphoinositides that are selectively enriched in other membrane compartments, such as PI4P within the golgi membrane or PIP2 at the resting plasma membrane. Thus, conveying lipid specificity to PH domains constitutes a key mechanism for spatially sequestering distinct effector proteins within cells. Recently, we unexpectedly identified the Ca2+-sensing protein Calmodulin as a protein ligand for the PIP3-binding PH domains of the Tec kinase, Itk and the Ser/Thr kinase Akt. CaM and PIP3 cooperate to enhance each other's binding to the Itk PH domain and was required in T cells for optimal production of the pro-inflammatory cytokine IL-17A. The ability of two separate PH domains to interact with CaM led us to investigate a potential for PH domains in general to interact with CaM. In this R03 application, we propose to systematically evaluate PH domains as CaM binding domains for the purpose of generating an online database that categorizes PH domains as lipid, CaM, and/or small GTPase binders. The reagents generated here will also be deposited into a DNA vector repository for general distribution.